Multiplex Real-Time PCR for Rapid β-Lactamase Gene Detection

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Solution Overview

Problem

Current methods for detecting antibiotic resistance, particularly β-lactamase enzymes, are limited in their ability to identify multiple gene families simultaneously and lack sensitivity, specificity, and automation, often relying on time-consuming agarose gel detection and conventional PCR.

Innovation Solution

A multiplex real-time PCR assay using specific primers and probes for various β-lactamase gene families, including metallo-β-lactamases, carbapenemases, and extended-spectrum β-lactamases, with optimized primer and probe concentrations, enabling rapid and comprehensive genotypic characterization of antibiotic resistance.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional PCR with agarose gel detection is used, then the assay can detect β-lactamase genes, but the resolution is poor and the process is time-consuming

Engineering Contradiction:

Improvedetection resolutionVSAvoidtest time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent replaces the mechanical/visual detection method of agarose gel electrophoresis with real-time fluorescent detection during PCR amplification. This substitution eliminates the need for gel casting, staining, and visual analysis, thereby improving resolution and reducing test time simultaneously.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent implements real-time monitoring of PCR amplification through fluorescent probes that continuously signal product formation during each cycle. This continuous detection eliminates the discontinuous nature of endpoint gel analysis, allowing rapid identification of amplification without requiring post-PCR processing steps.

Inventive Principle:

Principle #20Continuity of useful action

2Reliability

If conventional PCR is used, then the assay can identify resistance genes, but it lacks sensitivity and cannot be automated

Engineering Contradiction:

Improvedetection sensitivityVSAvoidautomation capability

Core Design Contradiction:

ReliabilityVSExtent of automation

Solution Approach 1:

The patent replaces manual gel analysis with automated real-time fluorescent detection systems. The fluorescent probes generate signals that can be automatically read and analyzed by instruments, enabling both high sensitivity through quantitative detection and full automation of the workflow.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The real-time PCR system provides continuous feedback on amplification status through fluorescent signal intensity, allowing automated interpretation of results based on threshold crossing or slope analysis. This feedback mechanism enables sensitive detection while maintaining automation through computer-controlled analysis.

Inventive Principle:

Principle #23Feedback

3Adaptability or versatility

If single primer sets are used for real-time PCR, then the assay can detect specific gene families, but the number of detectable gene families is limited

Engineering Contradiction:

Improvenumber of detectable gene familiesVSAvoidprimer set complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent divides the detection system into multiple independent primer-probe sets, each targeting specific β-lactamase gene families. This segmentation allows simultaneous detection of multiple gene types in parallel reactions, increasing versatility without requiring a single complex universal primer set.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent creates multi-functional primer-probe sets that can detect multiple gene families within a single reaction system. By designing primers that can amplify different targets or using universal amplification components, the system achieves broad detection capability while managing complexity through standardized reagent formulations.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The assay provides rapid, accurate, and sensitive detection of multiple β-lactamase gene families, enhancing surveillance, epidemiologic tracking, and patient therapy by improving clinical sensitivity and analytical specificity.

Implementation Method 1

multiplex real-time polymerase chain reaction

Methodology Applied

Scientific EffectPolymerase chain reaction:

Implementation Method 2

real-time polymerase chain reaction allows for monitoring of reaction products as they are formed

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS20250270657A1Detection of Antibiotic Resistance Genes

Publication Date: 2025.08.28 STRECK INC

AI summary

Assays and methods for detecting resistance to beta-lactam antibiotics including detection of multiple β-lactamase family specific gene targets by polymerase chain reaction or microarray. One or more kits including primers and/or probes for identification of β-lactamase genes selected from the group consisting of one or more of the following: MOX-like, FOX-like, ACC-like, ACT/MIR-like, CMY-2-like, DHA-like, CTX-M-14-like, CTX-M-15-like, VIM-like, NDM-like, IMP-like, KPC-like, and OXA-48-like, OXA-51-like, OXA-143-like, OXA-58-like, OXA-23-like, OXA-24/40-like, TEM-like, SHV-like, and GES-like. A kit may also include one or more primers and/or probes for the identification a non-beta lactamase gene family which confers antibiotic resistance, such as the MCR-1 gene.